156
Exp Oncol 2009
31, 3, 156–162
Experimental Oncology 31, 156–162, 2009 (September)
PLATINUM NANOCOLLOID-SUPPLEMENTED HYDROGENDISSOLVED WATER INHIBITS GROWTH OF HUMAN TONGUE
CARCINOMA CELLS PREFERENTIALLY OVER NORMAL CELLS
Y. Saitoh, Y. Yoshimura, K. Nakano, N. Miwa*
Cell-Death Control BioTechnology Laboratory, Faculty of Life and Environmental Sciences, Prefectural
University of Hiroshima, Shobara, Hiroshima 727-0023, Japan
Aim: Hydrogen-dissolved water (HD-water) or platinum nanocolloid (Pt-nc) has been individually expected as a new therapeutic
agent for oxidative stress-related diseases, whereas little is known about their combined effects on cancer, which were elucidated
in the present study. Methods: HD-water was prepared by microporous gas bubbling, and supplemented with Pt-nc consisting
of 0.003–1 ppm Pt and PVP polymers. Antioxidant activities were examined by 1, 1-diphenyl-picrylhydrazyl (DPPH)-radicalscavenging assay. Cytotoxic activities were examined by culturing of tumor and normal cell lines, respectively. Results: HD-water
accele­rated the Pt-nc-based DPPH-radical scavenging. Pt-nc-supplemented HD-water inhibited either colony formation efficiencies
or colony sizes of human tongue carcinoma cells HSC-4, in contrast to no effects of HD-water alone, Pt-nc alone or Pt-absent
PVP, but not appreciably inhibit normal human tongue epithelial-like cells DOK. Pt-nc-supplemented HD-water also suppressed
cell population growth of HSC-4 cells of near-confluence (at higher cell densities) in view of decreases in either cell numbers
or mitochondrial function, although less markedly than colony formation starting from a sparse-cell state (at lower cell densities).
Dissolved hydrogen, oxygen concentration or oxido-reduced potentials of HD-water was decreased, rather decreased or increased
by Pt-nc addition, respectively. Conclusions: Anti-cancer activity of Pt-nc-supplemented HD-water was shown by its preferential
cell-growth inhibition to human tongue carcinoma cells HSC-4 over normal human tongue cells DOK, and might be partly attributed
to HD-water-caused enhancement of Pt-nc-relevant antioxidant ability. Pt-nc-supplemented HD-water is expected as a novel
agent against human tongue cancers due to its cancer progression-repressive abilities.
Key Words: hydrogen-dissolved water, platinum nanocolloid, tumor-preferential repression, human tongue carcinoma, reactive
oxygen species, antioxidant.
It has been reported that human tumor cells produce reactive oxygen species (ROS) more abundantly
than non-transformed cell lines [1], and an elevated
oxidative stress has been found in many different types
of cancer cells [2]. In fact, many evidences suggest
that ROS are related to diverse abilities of cancer cells
which increase cell proliferation, DNA synthesis [3],
survival, cellular migration [4], invasion [5, 6], tumor
metastasis [7, 8] and angiogenesis [9]. Furthermore,
ROS within cells act as second messengers and play
important roles in intracellular signaling cascades
which induce and maintain the oncogenic phenotype
of cancer cells [1]. On the other hand, it is also known
that antioxidants can inhibit tumor cell proliferation,
which indicates an important role of ROS in mediating
the loss of growth control [10–12]. Thus, these findings
suggest the possibility that scavenging of ROS by antioxidant compounds or antioxidant materials can suppress the tumorigenic actions of ROS and the development of certain cancers.
Recently, many researchers have paid attention
to hydrogen as a novel antioxidant material, and reReceived: June 19, 2009.
*Correspondence:
Fax: 81824741754
E-mail: [email protected]
Abbreviations used: HD-water — Hydrogen-dissolved water; DH —
dissolved molecular hydrogen; DMEM — Dulbecco’s modified
Eagle’s medium; DO — dissolved oxygen; DPPH — 1, 1-diphenyl2-picrylhydrazyl; MEM — Eagle’s minimum essential medium;
ORP — oxido-reduced potential; PR — phenol red; Pt-nc — platinum nanocolloid; PVP — poly (N-vinyl-2-pyrrolidone); ROS — reactive oxygen species.
ported that hydrogen is quite effective for alleviating
oxidative stress. Inhalation of hydrogen gas markedly
suppressed focal ischemia and reperfusion-induced
brain injury in rat [13], reduces myocardial injury
by ischemia-reperfusion in rat [14], and suppresses
hepatic injury by ischemia-reperfusion in mouse [15].
Furthermore, many reports have been also demonstrated that aqueous solutions containing high concentrations of dissolved hydrogen (hydrogen-dissolved
water; HD-water), which were produced by electrolysis
or dissolved hydrogen under high pressure, show
the antioxidant activities and efficacies in various experimental disease models. It has been showed that
HD-water decreased superoxide anion radicals [16, 17],
hydrogen peroxide [18], hydroxyl radical [13, 18, 19],
and protects DNA [16, 20, 21], RNA [21] and protein
[21] from oxidative damages, and HD-water suppressed
antimycin A-induced cell death [13], alloxan-derived
ROS-induced pancreatic β-cell damage [22], tumorpreferential clonal-growth-inhibition, tumor invasion
[19] and tumor angiogenesis [23].
Moreover, it has been demonstrated that drin­
king HD-water decreased the ROS-induced urinary
secretion of 8-hydroxydeoxyguanosine and hepatic
formation of peroxidized lipid in rats [24], the hypoxiareoxygenation-induced ROS formation in vitamin
C-depleted mice [25], chronic physical restraint
stress-induced increase of oxidative stress mar­
kers (malondialdehyde and 4-hydroxy-2-nonenal
in the brain) [26] and ameliorate oxidative stressrelated diseases such as diabetes mellitus in mice [27,
28]. Furthermore, the efficacy of HD-water has been
Experimental Oncology 31, 156–162, 2009 (September)
demonstrated in clinical trials for the patients with
type 2 diabetes or impaired glucose tolerance [29]
and a hemodialysis-induced oxidative stress in endstage renal disease patients [30, 31].
On the other hand, platinum nanocolloids (Pt-nc)
are generally known to exhibit strong catalysis activity
because of their high surface reactivity, and it is also
known that Pt-nc have antioxidative effects [32]
and the ability to scavenge ROS, superoxide anion
radials (O2 • –), and hydrogen peroxide (H2O2) [33, 34].
Therefore, HD-water supplemented with Pt-nc is expected to be a strong new antioxidant and a candidate
for anti-cancer therapy. Indeed, it has been showed
that electrolyzed reduced water supplemented with
Pt-nc strongly prevents transformation of murine
Balb/c 3T3 cells [35].
In the present study, we investigated the anti-cancer effects of Pt-nc-supplemented HD-water for oral
tumor cells and its effects for normal cells.
MATERIALS AND METHODS
Hydrogen-dissolved water (HD-water). HD-water was prepared by microporous gas bubbling into
flowing water about 0.1 MPa at hydrogen pressure
of more than 0.6 MPa (HYDRO BATH; Hiroshima
Kasei, Hiroshima). Concentrations of dissolved molecular hydrogen (DH) in solution were measured
by using a DH meter (DH-35A, DKK-Toa, Tokyo),
and concentrations of dissolved oxygen (DO)
were measured by using a pH/DO meter (D-55,
Horiba, Kyoto), and oxido-reduced potential (ORP)
was measured by using an ORP meter (RM-20P,
DKK-Toa), respectively at 25 °C. In all experiments,
we used HD-water, which indicated each parameter
at 1.0–1.3 ppm (parts per million) of DH concentrations, -668 to -481 mV of ORP, and 7.0–7.8 of pH.
Hydrophilic colloid poly (N-vinyl-2-pyrrolidone) (PVP)entrapped Pt-nc, containing approximately 4% (wt%)
of 2-nm dia­meter platinum particles, which consist
of 8% (wt%) of PVP, was obtained (commercially
available) from Tanaka Kikinzoku Kogyo K. (Tokyo).
The production method was described in Laid-Open
Disclosure Public Patent Bulletin in Japan (333737,
1999) and Japan Patent (P4252151).
1,1-diphenyl-2-picrylhydrazyl (DPPH) radicalscavenging assay. DPPH radical-scavenging activi­
ty was investigated according to the previous report
[36] with minor modification. The solution containing
25 μM DPPH in 50% ethanol mixed with diverse species
of waters (purified water (control), purified water + Pt-nc,
purified water + PVP, HD-water and HD-water + Pt-nc),
and the absorbance of each mixture was scanned
at 400–700 nm after 30 s and 10 min, respectively, with
a spectrometer (U-2800, Hitachi, Tokyo).
Cell culture. Human tongue squamous carcinoma-derived cell line HSC-4 (RCB1902) was provided
by the Health Science Research Resource Bank (Osaka). Cells were cultured in Eagle’s minimum essential
medium (MEM; Nissui Seiyaku, Tokyo) supplemented
with 10% heat-inactivated fetal bovine serum (FBS, In-
157
vitrogen Corp., CA), and 2 mM L-glutamine [37]. Normal human tongue epithelial-like cells DOK were supplied by DS Pharma Biomedical Co., Ltd. (Osaka). Cells
were cultured in Dulbecco’s modified Eagle’s medium
(DMEM, Nissui Seiyaku) supplemented with 10% heatinactivated FBS, and 4 mM L-glutamine at 37 °C [38].
Both cell lines were cultured at 37 °C in a humidified
atmosphere of 95% air and 5% CO2. Culture medium
containing HD-water or purified water was prepared
by dilution of 10-fold concentrated culture medium
with each water, and was supplemented with Ptnc if necessary.
Colony formation assay. Cells were seeded
on a 24-well plate and allowed to attach to the substratum, and then the spent medium was exchanged
to fresh cell culture medium that was prepared with
HD-water or purified water, which was supplemented
with or without Pt-nc. After 3-day incubation, the colonies were stained and photographed, and the total
colony numbers per dish and cell numbers per colony
were evaluated under a microscope.
Evaluation of cell viability and cell number.
Cell viability was assessed based on mitochondrial
enzymatic conversion of WST-1 [2-(4-iodophenyl)-3(4-nitrophenyl)-5-(2.4-disulfophenyl)-2H-tetrazolium,
sodium salt] (Dojindo, Kumamoto) to yellowish formazan, which is indicative of the number of viable
cells. Cells were rinsed with phenol red (PR)-free
medium and then incubated for 3 h in PR-free medium containing 10% WST-1 at 37 °C. The absorbance
at 450 nm was measured with a mic­roplate reader
(FLUOstar OPTIMA, BMG Labtech, Offenburg). Cell
numbers were measured with a particle counter (CDA500, Sysmex, Kobe).
Statistical Analysis. The unpaired Student’s t-test
was used to evaluate the significance of differences
between groups, and the criterion of statistical significance was taken as P < 0.05.
RESULTS
Combined effects of HD-water and Pt-nc
on DPPH-radical-scavenging activity. Combined
effects of HD-water and Pt-nc on antioxidative actions
were investigated in a cell-free system by DPPHradical-scavenging assay (Fig. 1). The treatment
with purified water, purified water + PVP or HD-water
alone occurred scarce decreases in DPPH radicals
both for 30 s and 10 min. On the other hand, purified water + Pt-nc induced an appreciable decrease
in the radicals for 30 s, and a marked decrease
for 10 min. In contrast, HD-water + Pt-nc dramatically
decreased the radicals within 30 s. The antioxidant
activity of Pt-nc (1 ppm) is equal to that of Torolox
at 2.2 μM for 30 s, and or 5.3 μM for 10 min, respectively. Thus, combination of HD-water and Pt-nc exer­
ted a more rapid radical-scavenging activity than that
by treatment with purified water + Pt-nc. Therefore,
HD-water can accelerate or enhance the Pt-ncinduced DPPH-radical-scavenging activity.
158
Experimental Oncology 31, 156–162, 2009 (September)
Control
PVP
Pt-C
HDW
HDW + PVP
HDW + Pt-nc
30 s
0.12
0.1
0.08
0.06
a
HDW
+
0.14
450
500
550
600
Wavelength (nm)
650
700
Control
PVP
Pt-C
HDW
HDW + PVP
HDW + Pt-nc
10 min
0.12
0.1
0.08
0.02
0
400
450
500
550
600
Wavelength (nm)
650
700
Fig. 1. Combined effects of HD-water and Pt-nc on DPPH-radi­
cal-scavenging activity. After mixing a solution of DPPH radicals
and diverse species of waters: purified water (control), purified
water + PVP (2 ppm), purified water + Pt-nc (1 ppm), HD-water,
HD-water + PVP (2 ppm), and HD-water + Pt-nc (1 ppm). The absorbance was measured at 30 s and 10 min. DPPH gives a strong
absorption around 520 nm. HPW — HD-water
Repressive effects of concomitant administration with HD-water and Pt-nc on colony
formation of tongue carcinoma cells HSC-4.
It was investigated whether concomitant administration with HD-water and Pt-nc could repress colony
formation of human tongue carcinoma cells HSC-4.
The colony number was not affected by purified
water at any concentrations of Pt-nc, but it was significantly reduced by concomitant administration
with HD-water and Pt-nc increasingly in a manner
dependent on Pt-nc doses (Fig. 2, a, b). The repressive effects were nearly equal to those between
two modes for Pt-nc-administration timings (“0 h”
and “24 h”). Furthermore, a cell number per co­lony
was substantially reduced upon the concomitant administration with HD-water and Pt-nc as compared
with other treatments (Fig. 2, c). These results indicate
that the concomitant administration with HD-water and
Pt-nc exerted repressive effects against both colony
formation and cell proliferation in HSC-4 cells.
Effects of PVP on colony formation of HSC-4
cells. The effects of PVP, which is a entrapping
and protective polymer agent for Pt to form colloidal
particles, on colony formation of HSC-4 cells were
examined. The colony number was not affected
by any concentrations of PVP regardless of combination with HD-water (Fig. 3). The results suggest
that the Pt itself is quite important for the anti-cancer
effects of concomitant admi­nistration with HD-water
and Pt-nc.
**
**
**
400
350
300
250
200
150
100
50
0
0.003
**
Pt-nc
1 ppm
(Pt equivalent)
–
PW
0.003
0.01
0.03
0.1
Pt-nc (ppm; Pt equivalent)
PW
HDW
0.3
**
1
c
Average 12.6
20
10
0
30
Average 12.2
20
10
20
Average 11.1
10
0
30
HDW
+
1
**
30
0
30
–
0.3
**
0
+
0.01
0.03
0.1
Pt-nc (ppm; Pt equivalent)
24 hr
**
Frequency
0.04
PW
b
HDW
*
0
0.06
50 µm
0 hr
Frequency
400
500 µm
400
350
300
250
200
150
100
50
0
Frequency
0
Colony number/well
0.02
Frequency
0.04
Absorbance of DPPH radicals
PW
Pt-nc
1 ppm
–
Colony number/well
Absorbance of DPPH radicals
0.14
Average 3.8
20
10
0
1 3 5 7 9 11 13 15 17 19 21 23 25 27 29
Cell number per colony
Fig. 2. Effects of concomitant administration with HD-water
and Pt-nc on colony formation of human tongue carcinoma cells
HSC-4. Cells were seeded into Pt-nc (0–1 ppm)-containing cell
culture medium prepared with HD-water or purified water at a density of 500 cells per well. After 3-day incubation, the colonies were
stained and photographed. a, Morphological aspects of treated
cells. The white and black scale bars indicate 500 µm and 50 µm,
respectively. b, Total colony numbers were evaluated by counting
the colonies which consisted of more than 8 cells per colony. Cells
were treated with once at only 0 h (when they were seeded) (the upper figure) or twice at 0 h and 24 h after seeding (the lower figure),
respectively. Significantly different from purified water: *P < 0.05,
**P < 0.01. c, The distribution of cell numbers per colony was also
evaluated for 90 colonies under a microscope. PW — purified water;
HDW — HD-water; Average — an average cell numbers per colony
PW
HDW
200
150
100
50
0
0
0.006
0.02
0.06
0.2
PVP (ppm)
0.6
2
Fig. 3. Effects of PVP on colony formation of human tongue
carcinoma cells HSC-4. Cells were seeded into PVP-containing
cell culture medium prepared with HD-water or purified water
at a density of 500 cells per well. After 3 days of incubation,
the colonies were stained, and total colony numbers were
evaluated by counting the colonies which consisted of more than
8 cells per colony. PW — purified water; HDW — HD-water
Repressive effects of concomitant admi­
nistration with HD-water and Pt-nc on colony
formation of carcinoma cells preferentially over
normal cells. The effects of concomitant administration with HD-water and Pt-nc on colony formation
of normal human tongue epithelial-like cells DOK were
investigated. As shown in Fig. 4, the colony number
was decreased to less than 5% of the maximum
for HSC-4 cells by the concomitant administration with
HD-water and Pt-nc at 0.3 ppm, whereas the number
was scarcely affected for DOK cells. Thus, HD-water
did not or scarcely affect the clonal growth of normal
cells, but preferentially repressed proliferation of carcinoma cells even regardless of similar cell growth rates
of DOK cells (a doubling time of approximately 24 h)
and HSC-4 cells (approximately 24 h).
Effects of concomitant administration with
HD-water and Pt-nc on cell growth of HSC-4 cells.
In addition to effects on clonal growth, the inhibitory
effects of HD-water on tumor population growth were
also investigated. The cell-growth of HSC-4 cells
was significantly inhibited by the concomitant admi­
nistration with HD-water and Pt-nc as compared with
purified water + Pt-nc (Fig. 5). The repressive effects
were nearly equal to those between two modes for
Pt-nc-administration timings (4 h and 24 h), and these
results showed similar tendencies by two methods
for evaluation (cell viability estimated by WST-1 assay
and cell number by counting with a particle counter).
Temporal changes of DH, ORP and pH in cell
culture medium prepared with purified water
or HD-water. The temporal changes of DH, ORP,
DO and pH in cell culture medium which was prepared with purified water or HD-water were examined, and effects of Pt-nc on their values were also
investigated (Fig. 6). Although HD-water showed
a high DH and low ORP, the DH value was immediately lowered, by mixing into the culture medium
and the subsequent filtration with a 0.10-μm-pore
filter, from approximately 1.0 to 0.3 ppm. Moreover,
the DH values of culture medium that was produced
by HD-water, were much higher than by purified water,
and decreased in a time-dependent manner, and they
finally reached a level equal to that of purified water
at 4 h after preparation. On the other hand, the addition of Pt-nc decreased the DH values in a dosedependent manner more markedly than for HD-water
alone. The addition of Pt-nc increased the ORP values
in a dose-dependent manner more markedly than
for HD-water alone. The DO values of HD-water
were lower than those of purified water, but the effects of Pt-nc addition were not clear. The pH values
scarcely changed and kept physiological levels around
pH 7.3–7.9 for 4 h.
HSC-4
a
DOK
PW
HDW
PW + Pt-nc
(0.3 ppm)
(Pt equivalent)
HDW + Pt-nc
(0.3 ppm)
(Pt equivalent)
Colony number/well
450
400
350
300
250
159
500
450
400
350
300
250
200
150
100
50
0
HSC-4
b
**
**
**
0
Colony number/well
Colony number/well
Experimental Oncology 31, 156–162, 2009 (September)
500
450
400
350
300
250
200
150
100
50
0
0.01
0.03
0.1
0.3
Pt-nc (ppm; Pt equivalent)
1
DOK
PW
HDW
*
**
0
0.01
0.03
0.1
0.3
Pt-nc (ppm; Pt equivalent)
1
Fig. 4. Differential effects of HD-water on colony formation
of human tongue carcinoma cells HSC-4 and normal human tongue epi­thelial-like cells DOK. Cells were seeded into
Pt‑nc containing cell culture medium prepared with HD-water
or purified water at a density of 500 cells per well. After 3-days
of incubation, the colonies were stained, photographed. a, Morphological aspects of treated cells. The white and black scale
bars indicate 500 µm and 50 µm, respectively. b, Total colony
numbers were evaluated by counting the colonies which consisted of more than 8 cells per colony. Significantly different
from 0 ppm of PW or HDW, respectively: *P < 0.05, **P < 0.01.
PW — purified water; HDW — HD-water
Experimental Oncology 31, 156–162, 2009 (September)
4h
**
30 000
*
**
60
**
40
10 000
20
0
0
0
Cell viability:
0.01 0.03
0.1
0.3
Pt-nc (ppm; Pt equivalent)
PW
HDW
PW cell number
HDW cell number
**
30 000
20 000
60
40
10 000
20
0
-0.05
0.01 0.03
0.1
0.3
1
Pt-nc (ppm; Pt equivalent)
Cell number: PW
HDW PW cell number HDW cell number
In the present study, we investigated the combined
effects of HD-water and Pt-nc on human oral tumor cell
growth, and their effects on normal cells derived in common from human tongue. Our data showed the repressive
effects against both colony formation and cell proliferation
in HSC-4 cells were exerted by the combination of HD-water and Pt-nc, but not purified water alone, purified water +
Pt-nc, HD-water alone or HD-water + PVP (see Fig. 2, 3).
These results suggest that Pt-nc-supplemented HD-water
plays a quite important role for the anti-cancer effects.
Furthermore, the treatment of Pt-nc-supplemented
HD-water exerted the preferred prevention against colony
formation and cell proliferation of carcinoma cells over
normal cells (see Fig. 4). In addition to inhibitory effects
of Pt-nc-supplemented HD-water on colony formation
which of carcinoma cells, correspond to “clonal growth”
immediately after formation of a single cancerous or mutated cell, Pt-nc-supplemented HD-water was furthermore
demonstrated to inhibit also “massive growth” as a cancer
cell population as shown in Fig. 5, which corresponds
to an early stage of cancer progression. Though the inhibitory effects on “massive growth” were comparatively
weaker than those on “clonal growth” (see Fig. 2–5), these
results suggest that the decrease in anti-cancer effects
is due to either the enhancement of cellular resistance
against hydorogen and Pt-nc or reduction of the accessibilities of hydorogen and Pt-nc because of prevention
2
Time (hr)
3
4
0
1
2
Time (hr)
3
4
0
1
2
Time (hr)
3
4
0
1
2
Time (hr)
3
7
6
5
4
3
200
ORP (mV)
100
0
-100
-200
-300
-400
-500
9.0
8.5
8.0
pH
DISCUSSION
1
8
0
Fig. 5. Effects of concomitant administration with HD-water and
Pt-nc on cell growth of human tongue carcinoma cells HSC-4.
Cells were seeded at a density of 1.2 × 104 cells per well in a 24well plate. After 4 or 24-h incubation, the medium was exchanged
to Pt-nc containing cell culture medium prepared with HD-water
or purified water. After 24-h incubation, the cell-growth ratio
was assessed by the WST-1 assay and counting cell number,
respectively. Significantly different from purified water: *P < 0.05,
**P < 0.01. PW — purified water; HDW — HD-water
0
9
0
0
0.2
0.15
0.1
40 000
**
0.25
0.05
100
80
HDW + Pt-nc (0.03 ppm; Pt equivalent)
HDW + Pt-nc (0.3 ppm; Pt equivalent)
PW
HDW
0.3
1
24h
120
Cell viability (%)
20 000
0.35
DO (ppm)
**
80
by the decrease in cellular superificial area accompanied
with cellular growth and the subsequent increase in cell
density.
Cell number (cells/well)
Cell viability (%)
100
40 000
Cell number (cells/well)
120
DH (ppm)
160
7.5
7.0
6.5
6.0
4
Fig. 6. Temporal changes of DH, ORP, DO and pH in cell culture medium prepared with HD-water or purified water and their dependency
on the presence or absence of Pt-nc. After preparation of each medium, they were incubated at 37 °C in a humidified atmosphere of 95%
air and 5% CO2. Then, the parameters were measured at the indicated time points. PW — purified water; HDW — HD-water
The anti-cancer effects of Pt-nc-supplemented HDwater are suspected to be due to their antioxidant activity,
because ROS are related to diverse abilities of cancer cells
[1–12]. This is supported by the results that HD-water
Experimental Oncology 31, 156–162, 2009 (September)
could accelerate or enhance the Pt-nc-induced radical
scavenging activity (see Fig. 1). Many studies suggested
that HD-water possessed the antioxidant activity [13,
16–19], but our results showed that anti-cancer effects
were insufficient only with HD-water. Two reasons are able
to be considered as the causes of these results. As the first
reason, the DH values of HD-water in our experiments were
low as compared with those in other reports. In our experiments, the DH values markedly decreased immediately
after mixing with cell culture medium, and they are ra­
pidly decreased in a time-dependent manner as shown
in Fig. 6. It was demonstrated that DH levels are related with
an antioxidant effect [13]. Consequently, the treatment
with HD-water alone could not show an excellent antioxidant activity, and could not necessarily exert sufficient
anti-cancer effects in our experiments. And the second
reason is the co-existence of Pt. There is a possibility that
the nature of DH-water may vary according to diverse
principle in the methods for the production of HD-water
(by electrolysis, dissolved hydrogen under high pressure,
or magnesium addition to water). It was assumed that
a trace of Pt nanoclusters existed in electrolyzed reduced
water by releasing from the Pt-coated electrodes during
electrolysis [39]. Since it is well known that metal clusters
(such as Pt) can act as catalysts for reductive reactions
in aqueous solutions, it is presumed that the HD-water produced by electrolysis contains a trace amount of Pt which
helps the antioxidant activity of HD-water. However,
there is a few possibility that Pt exists in the HD-water
produced by hydrogen bubbling. Therefore, it seems that
the HD-water in our experiments could not exert the anticancer effects by itself alone. Taken together, although
HD-water alone could not exert sufficient anti-cancer
effects as compared with previous reports, our results
might be able to be explained by the DH values of HDwater or the differences in the methods for the production
of HD-water. These hypotheses coincide with our results
that the anti-cancer effects were achieved by the coexistence of Pt-nc in spite of low DH values. These results
also showed a possibility that the addition of Pt-nc into
HD-water can overcome its instability as the weak points,
and can enhance its antioxidant activities. Consequently,
the DH-water has still potentials for the improvement
and augmentation of the anti-cancer activities by finding
the way to elevate and keep DH values at higher levels.
Synthesized Pt nanoparticles (nps) of 2 nm or less
at a diameter can disperse in aqueous solutions for a long
time and function as efficient catalysts to convert hydrogen
molecules to atomic hydrogens on the surface of Pt metal
[35, 40]. Some reports assumed that the atomic hydrogen scavenge ROS, and therefore, we also postulate that
atomic hydrogen, which generated on the surface of Pt,
may be related with anti-cancer effects in our experiments. The decrease in DH values by the addition of Pt-nc
(see Fig. 6) might be attributed to the catalytic action
of Pt. Moreover, we revealed that palladium-PVP, which
is another catalyst for hydrogen molecules, also showed
the similar anti-cancer effects (data not shown). These
assumptions are consistent with our results that the anti-
161
cancer effects were endowed by HD-water supplemented
with Pt-nc but not by HD-water or Pt-nc individually.
Recently, it has been showed that electrolyzed reduced water supplemented with Pt nanoparticles strongly prevents transformation of murine Balb/c 3T3 cells
[35]. In this report, it was postulated that Pt nanoparticles
and hydrogen molecules were incorporated into cells,
molecular hydrogen is converted to atomic hydrogen
on the surface of Pt nanoparticles, and atomic hydrogen
or electrons on the surface of Pt nanoparticles scavenge intracellular ROS. Moreover, they suggested that
the decrease in ROS generation changed biochemical
signaling pathways via redox-sensitive molecules like
AP-1 and NF-κB, interrupting the cascade pathway,
which may promote cell transformation [41]. Their
hypothesis is considered to be applied to our results
that anti-cancer effects were achieved by the addition
of Pt-nc into HD-water. Therefore, we presume that
the anti-cancer effects of Pt-nc-supplemented HD-water
on human cancer cells are achieved through its antioxidant activity. Moreover, augmentation of this antioxidant
activity of Pt-nc-supplemented HD-water may become
a reinforcing tool useful for certain types of anticancer
therapy such as radiotherapy or certain anti-cancer
drugs which are concerned with ROS-induced cytotoxicities [42]. On the other hand, although we have
not compare HD-water plus Pt-nc with some traditional
Pt drugs, we think that the anticancer mechanism of HDwater with Pt-nc is different from the traditional Pt drugs
such as DNA synthesis inhibitor cisplatin, because it is reported that cisplatin induces toxic affects via generation
of reactive oxygen species [42]. However, we also think
that a comparison with traditional Pt drugs is interesting
and is worthwhile to try in a future experiment.
The present study demonstrates that Pt-nc-supple­
mented HD-water exerts a more prompt antioxidant action and preferentially inhibited clonal-growth of human
tongue carcinoma cells over that of the corresponding
normal cells. Therefore, Pt-nc-supplemented HD-water
is expected to become a useful tool for clinical application to anti-cancer therapy, and the further in vivo stu­
dies are necessary for clinical applications.
ACKNOWLEDGEMENTS
The authors thank Ms. Haruko Mimura and Ms. Hiroe Masaki for their technical assistance.
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